Training aid

ABSTRACT

Provided herein are methods of making a training aid for detecting a biohazard, and related devices and methods of using the device, including a canine training aid. The devices are made by positioning a polymer layer in proximity and physically separated from a biological material, so that volatile organic compounds contact and bind or infuse the polymer layer. The device is made render-safe by inactivating the infused polymer layer, such as by heating to a temperature sufficient to inactivate biological agents on or in the analyte-infused polymer layer. The device can be stored in a substantially air-tight configuration for subsequent use in training, such as canine and/or or artificial detectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Nos. 62/926,725, filed Oct. 28, 2019 and 63/036,738,filed Jun. 9, 2020, which are hereby incorporated by reference in theirentirety.

STATEMENT OF GOVERNMENT INTEREST

The inventions described herein were invented by employees of the UnitedStates Government and thus, may be manufactured and used by or for theU.S. Government for governmental purposes without the payment ofroyalties.

BACKGROUND OF THE INVENTION

Law enforcement has a need for field detection of biological agents andtoxins that may be weaponized, including those that are characterized asBiological Select Agents and Toxins (BSAT). The U.S. Government hasdetermined that BSAT have the potential to pose a severe threat to thepublic, animal and/or plant health, or to animal or plant products andthus possession, use, and transfer are restricted to select agentlaboratories that are registered with the U.S. Centers for DiseaseControl and Prevention (CDC) or the U.S. Department of Agriculture(USDA). Registered facilities are regulated for compliance with a numberof policies and procedures designed to maintain the security of thesematerials. Furthermore, there is a need in the art for reliable andefficient detection of biological agents that are associated with apandemic, or at risk of developing into a pandemic, including thoseagents that affect public health, agriculture or animal health. Providedherein are methods of making training aids, and related methods of usingsuch aids, and various training aids, including in the context of animal(including canine) training and device calibration, that address the endneed for field detection of such agents in a safe, reliable andcost-effective manner.

Canine detection is currently the most reliable means of field detectionof biological agents and toxins, surpassing current technologicalcapabilities of handheld instruments. Canines are used as a screeningtool for law enforcement to help direct forensic personnel to locationsto be processed so that confirmatory testing can be conducted in alaboratory. Canines who meet the criteria for detection work aretypically taught in a secure training facility to detect hazardoussubstances such as explosives, narcotics, and chemicals by using actualmaterial combined with a physical barrier. This training and maintenancecannot take place in the canine's operational environment, such as anactive airport or train station, because these materials are highlyvolatile and/or dangerous to human health. Studies show that canines whoare taught in this environment may be less vigilant in their operationalwork environment due to not regularly encountering, and thus beingrewarded less for finding, a target.

BSAT cannot be used in the field for training due to the inherentdangers presented by the B SAT to human, animal, or plant health duringpreparation, transportation, and training. The present inventionaddresses this problem by developing a training aid to capture thevolatile organic compounds (VOCs) of a biological agent or toxin andthen rendering the training aid sterile by performing a sterilizationstep on the training aid, including for microbial VOCs (MVOCs) generatedby a bacteria, virus and related infection thereof.

Vapor capture and release systems are generally known in the art. U.S.Pat. Pub. No. 2014/0021270 (MacCrehan et al.), for example, describesstoring and releasing target agents with a polymeric substrate. Thatsystem, however, is fundamentally incompatible for safe and reliablecanine training with biological agents or toxins generated bymicroorganisms as there is a risk that active agents remain associatedwith those systems. In other words, those systems are not fairlydescribed as having been rendered safe. The systems and methods providedherein, in contrast, ensure that the canine training aid is renderedsafe so that there is no risk from biological agents or toxinsassociated with training canines, without adversely impacting caninetraining efficacy.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods, and related devices, that have beenrendered safe for device calibration and animal training, such as caninetraining, to detect biological agents, such as bacteria and/or viruses,and more generally, toxins. This is achieved by infusing a polymer withVOCs released by a material to-be-detected (“target agent”) by thecanine (e.g., a biological agent or toxin) and then heating theVOC-infused polymer to provide a rendered-safe training aid. The heatingensures that the VOC-infused polymer is sterilized so that there is norisk to the public, trainer, canine or the surrounding environmentduring training. Furthermore, the canine can be kept engaged duringoperational activity by selective use and placement of the training aidfor periodic canine reward. This ensures the canine remains activelyengaged for longer time periods, and has a number of functionalbenefits, including increasing effectiveness and detection, increasingeffective time on-site and overall vigilance. Without such positiverewards, even the best of canines are prone to boredom, associateddistraction, and decreased effectiveness.

The methods are based, at least in part, on the discovery that thecanine training aid can be effectively sterilized without adverselyimpacting the VOCs “stored” by the polymer in the training aid withrespect to canine detection. Examples of sterilizing include viaheating, such as to a temperature that is greater than about 125° C. fora time period sufficient to inactivate agents, including biologicalagents, that may reside on or in the polymer and that pose a health orenvironmental risk. Generally, as the temperature increases, the timeperiod required to achieve sterilization decreases. For example, at anelevated temperature of 140° C., the time the training aid is exposed tothat temperature may be in the approximately 1 hour range. For atemperature of 125° C., the time may be greater than 1 hour, such as 2hours or more. The time, temperature and/or pressure are agent-specific,and are determined by scientifically valid methodology, includingpublished methods used by laboratory subject matter experts. Forexample, LD50 to LD100 assays, where organism viability is empiricallydetermined as a function of time, temperature and/or pressure.

The invention provided herein is compatible with a wide range oftemperatures, so long as the temperature is sufficiently high to achievea render-safe aid, but not so high that the polymer-stored VOCs areadversely impacted to make the canine training ineffective. For example,the maximum temperature may be selected to be below 200° C., so that therange of maximum temperature for the heating of the VOCs-infused polymeris between about 130° C. and 150° C. The temperature is preferablyselected to avoid undue loss of VOCs from the polymer and long-termdamage to the polymer integrity, including a PDMS substrate.

The heating may be provided by autoclaving the VOCs-infused polymer.This provides increased sterilization capability through control of bothtemperature and pressure, as well as steaming and duration. Pre- andpost-sterilization studies show that the sterilization process does notadversely impact the canine's ability to detect the underlying agent.This allows canines to be trained on actual odor of a target biologicalagent or toxin without exposing the canine or handler to the targetitself. Of course, while the examples provided herein are directed tocanine training, the methods and devices described herein may be used totrain any animal or machine to identify the associated biologicalmaterial and/or toxin because the training aid is rendered-safe and hasbeen established to be reliably associated with the underlyingbiological agent and/or toxin.

More generally, the methods provided herein rely on inactivating agentsto ensure the resultant training aid is rendered-safe, withapplicability for biological agents and/or toxins. Accordingly, variousother inactivation protocols may be used in the method, besidestemperature increase. For example, besides heating by steam heating(e.g., autoclave) or dry heating, electromagnetic radiation of aspecific wavelength may be used, including wavelengths where damage isdone to biological material such as DNA and RNA and other microorganismcomponents, such as ionizing radiation or UV radiation. The wavelengthis selected to have a penetration depth in the polymer that is greaterthan or equal to the maximum depth of the biological agent in thepolymer.

Filtering may also be used to ensure that no agent is able to come intocontact with the polymer, including toxin or a biological agent. Thefiltering may be prior to the capturing (e.g., “charging”), such as byfiltering culture media used to “culture” the biological agent that is amicroorganism, such as a bacteria or a virus. For bacteria, the filtergenerally corresponds to a 0.2 μm size filter. Filtering generally isnot used for a virus because the size of the virus is so small thateffective filtering can be challenging.

Provided herein is a method of making a canine training aid fordetecting a biohazard. The method may comprise the steps of: providing amaterial that releases one or more analytes; positioning a polymer layerin proximity and physically separated from the material, wherein the oneor more analytes are capable of release from the material and contactwith the polymer layer; capturing the one or more analytes that contactthe polymer layer to obtain an analyte-infused polymer layer; heatingthe analyte-infused polymer layer to a temperature sufficient toinactivate agents on or in the analyte-infused polymer layer and torender-safe the analyte-infused polymer layer; and storing therender-safe analyte-infused polymer layer in a substantially air-tightcanister with a removable lid for subsequent use in canine training. Thebiohazard may correspond to a biological material that is amicroorganism, such as a bacteria or virus, or a toxin. In this manner,canines can be efficiently, safely and routinely trained to detect thepresence of one or more bacterial and/or viral agents. As newly emergentmicroorganisms or toxins become a risk, canines are able to be rapidlytrained and deployed using the training-aids described herein that arematched to the newly emergent target. This has immediate practicalapplications for efficient field detection of infectious agents ortoxins, including in high-risk areas such as airports, schools andbusinesses, such as theaters, sporting events, and concerts, as well asat borders and ports of entry for detection of microorganisms in foodand agriculture products, and domestic or wild animal populations. Thematerial can be a biological material and the agent can be a biologicalagent.

The methods and devices provided herein may be used to detect abiological agent and/or a toxin. Although a focus of the examplessection relates to a biological agent, the devices and methods canrelate, in a similar fashion, to toxins that can impact health andsafety of persons, animals, plants and the environment. The commonmechanism for the various agents are that they release VOCs that arecaptured in the polymer, and the VOCs-infused polymer is subsequentlysubject to a render-safe challenge to ensure the infused polymer is safefor subsequent handling and use without adversely impacting training,such as canine training. The render-safe step is referred to as an“inactivating” step that degrades or otherwise impacts the underlyingagent without adversely impacting the infused polymer's ability forsubsequent training.

The polymer layer may be positioned in an inner volume defined by ashallow and seamless canister, including wherein the removable lid isconfigured to cover the canister inner volume having the polymer layerin a substantially air-tight configuration.

The method may further comprise the step of heating the polymer layerand a canister before the positioning step to remove unwanted volatileorganic compounds associated with the polymer or the canister. In thismanner, even before the polymer layer is introduced to the biologicalagent or toxin the polymer layer and related canister and lid, have beentreated to remove at least substantial amounts of inherent VOCs thatcould otherwise interfere with the attendant reliability (e.g.,sensitivity and/or specificity) achieved with the training aid.

The heating step may further comprise heating the analyte-infusedpolymer layer and canister, including separately or with the polymer orpre-polymer already positioned in the canister.

The heating step may comprise autoclaving the polymer layer to anelevated temperature and pressure to sterilize the analyte-infusedpolymer layer. The autoclaving may comprise steam-sterilization of theanalyte-infused polymer layer.

Methods described herein are compatible with a range of heatingregimens, depending on the application of interest, including tailoredto the biological material of interest. The hearting may compriseintroducing the analyte-infused polymer layer to a temperature that isbetween 125° C. and 155° C. for a time period of between 0.5 hours and1.5 hours.

After the heating and storing step, the training aid is used to train acanine to detect the biological agent at a sensitivity of at least 85%and/or a specificity of at least 90%.

The method may, prior to the polymer layer positioned in the innervolume, further comprise the steps of: separating the lid from thecanister; baking the separated lid and canister at 140° C.-160° C. forat least three hours; cooling the separated lid and canister; andsealing the lid to the canister until the canister is ready to receive apre-polymer or the polymer layer.

The method is compatible with a range of polymers. For example, thepolymer layer may comprise polydimethylsiloxane (PDMS) or is PDMS. ThePDMS layer may have a depth that is greater than or equal to 2 mm andless than or equal to a canister inner volume depth. Any polymer may beused that is able to capture one or more analytes from the biologicalmaterial and withstand the inactivating step, such as by a heating step.

Any of the methods may further comprise the step of removing unwantedpolymer impurities by heating the polymer layer to at least 125° C. forat least one hour.

The method may further comprise the steps of: providing at least onemicroorganism and/or toxin that releases an analyte comprising avolatile organic compound (VOC); suspending the polymer layer over thebiological agent for a time period sufficient to contact the releasedVOC with the polymer layer; thereby loading the polymer with the VOC.

The agent may be a microorganism. The microorganism may be selected fromthe group consisting of: a bacteria; a virus; fungi; a prion; or anycomponent or combination thereof. Accordingly, the method may be fortraining to detect a bacterium. The method may be for training to detecta virus. The method may be for training to detect a toxin or anychemical posing a risk, so long as the toxin or chemical may be reliablydeactivated or destroyed by heating without permanently destroying thepolymer-infused material used to conduct the training in a render-safemanner.

The agent may be a toxin. The toxin may be selected from the groupconsisting of: a plant toxin (e.g., ricin, abrin); a bacterial toxin(e.g., botulinum, Shiga); a marine toxin (e.g., saxitoxin); a fungaltoxin (e.g., mycotoxin); and a combination thereof. The agent may be achemical. The inactivating of the toxins and/or chemicals may be by oneor more of: autoclaving (steam-heating) and/or filtration.

The method may be for training to detect two or more biohazards, such asa combination of biohazards. This can be accomplished by capturing witha single polymer layer analytes associated with at least two biohazards.Alternatively, a canine can be trained with two or more training aids,where each training aid has analytes from a unique biological material.In these manners, a single canine can detect presence of at least twodifferent biological materials, such as coronavirus or influenzainfection in an individual.

The method may be used to train an animal. The method may be used toimprove machine detection, including calibration and testing whileavoiding use of the actual biohazard itself.

The methods provided herein have a number of other useful benefits. Forexample, the methods ensure the resultant device can have a very longshelf-life, wherein shelf-life is indicative of the time elapsed sincethe training aid and wherein the training aid remains useful forreliable training. For example, the lid and canister in a storedconfiguration can provide a shelf-life for a canine-training applicationof at least six-months. This is a reflection of the reliable charging ofthe polymer with VOC and the chemical kinetics of VOCs within the closedcanister.

The methods are particularly suited to a biological agent that is amicroorganism, such as a microorganism that impacts veterinary healthand/or public health. For example, the microorganism may be an animaldisease virus, including a foreign animal disease virus of interest to agovernment agency (e.g., USDA or FDA) such as African swine fever virus,a coronavirus, or an influenza virus.

Also provided herein are render-safe training aids, including aids madeby any of the methods described herein.

A render-safe training aid may comprise: a canister having a bottomsurface and one or more side walls to define an interior volume with anopen upper surface (e.g., bottom surface faces the absent upper surface,and the canister interior can be exposed to the environment. A removablelid is removably connected to the canister by one or more side walls;and a sterilized VOC-infused polymer positioned supported by thecanister bottom surface. Of course, as the fundamental platform of theinstant technology is the render-safe platform, the training aid iscompatible with any number of geometries or configurations, includingcloseable vents that provide access to the canister interior, therebyavoiding the need for a canister having separate lid and base pieces.

Also provided herein are canine training methods using any of therender-safe training aids described herein, or made by any of themethods described herein.

The training aid may be reusable. For example, reusing of therender-safe training aid may occur over a time period encompassing atleast 10 different times for the same canine, a different canine, or acombination of the same and different canines. Multiple uses can beaccomplished by simply storing the charged render-safe polymer in aclosed canister until the next time the aid is required. This caneffectively act to prevent or at least substantially minimize loss ofVOCs from the polymer when stored.

Without wishing to be bound by any particular theory, there may bediscussion herein of beliefs or understandings of underlying principlesrelating to the devices and methods disclosed herein. It is recognizedthat regardless of the ultimate correctness of any mechanisticexplanation or hypothesis, an embodiment of the invention cannonetheless be operative and useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a method of making a training aid,including a canine training aid.

FIG. 2 is a process flow illustration of the details of the trainingaid, and related training aid stored and deployed configuration. Thedashed lines indicate that the training aid is optionally reusable for anumber of deployments, wherein between deployed training aid use, thesterilized and VOC-charged (e.g., infused) polymer is positioned in aclosed canister.

FIG. 3. Distractor and Target Summary: Relationship and pertinentplasmid comparison between target and distractor strains. Data shown iscross-referenced information between multiple sources to includeBazinet, et al., “Pan-genome and phylogeny of Bacillus cereus sensulato.” (Bazinet 2017); Genome Taxonomy Databasehttp://gtdb.ecogenomic.org/; NCBI Genomehttps://www.ncbi.nlm.nih.gov/genome/; and origin data from publicationsby Radnedge et al., and Kolsto et al. (Radnedge, Agron et al. 2003,Kolsto, Tourasse et al. 2009).

FIGS. 4A-4B are similar to FIG. 1, but illustrate the method iscompatible with a range of inactivation processes to obtain arender-safe training aid. FIG. 4A illustrates the heating of the chargedpolymer is compatible with other processes that inactivate the chargedpolymer for a render-safe polymer. FIG. 4B illustrates an inactivatingstep may occur before the charging, on the biological material itselfprior to introduction to the polymer. In this manner, the chargedpolymer may be directly stored after changing, thereby avoidinginactivation step post-charging. For extra safety, both pre- and apost-inactivation steps, or multiple post-inactivation processes may beused.

DETAILED DESCRIPTION OF THE INVENTION

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. Referring tothe drawings, like numerals indicate like elements and the same numberappearing in more than one drawing refers to the same element. Thefollowing definitions are provided to clarify their specific use in thecontext of the invention.

As used herein, “biological material” refers to a substance that canimpact biological well-being, including a bacteria, virus, fungus,prions, toxins, or related material, including any of those that may becharacterized as BSAT and/or of potential risk of generating a diseaseoutbreak impacting public or animal health, including pandemicoutbreaks. Particularly relevant are those that can be used as a weaponof mass destruction, are part of a pandemic including SARS-CoV-2(causative agent of COVID-19), foreign animal diseases (e.g., Africanswine fever virus), and/or are identified as an emerging threat. Theemerging threat can be directly to humans, or to an animal or a plant,including animals or plants of commercial value. The biological materialmay be in any of a wide variety of forms, including in a cell culture, acultured layer on a substrate, a suspension in a biological fluid, atissue sample, or the like. The cell culture may include a biologicalmaterial that is a microorganism such as a bacteria or a virus,including SARS-CoV-2 or other coronaviruses, African swine fever virusor other foreign animal diseases, or influenza viruses. For viralbiological agents, the virus includes those that are capable ofinfecting a cell culture and releasing odors that can be used to traincanines with any of the training aids provided herein. Similarly, thebiological agent may correspond to samples obtained from infectedindividuals (human, animal or plant) that are then exposed to thetraining aid polymer layer. A “biological agent” may correspond to the“biological material”, or it may be different. For example, the polymermay be exposed to a biological material that is a cell culture thatcontains biological cells infected with a biological agent that is avirus. The biological agent in this context, is viable virus, so thatthe methods and devices provided herein relate to ensuring anybiological agent is inactivated. Similarly, for a bacterial application,the biological material may be a combination of bacteria and anothermaterial, such as an organic living material, or any other substancedepending on the application of interest, such as soil, dirt, foodstuff,agricultural products, and the like, with the biological agentcorresponding to the bacteria.

“Toxin”, similar to biological agent, refers to a substance that canhave a biological impact but that is not itself capable of reproducing.Instead, it can be produced by a living organism and acts as a poison toanother living organism, including humans, animals or plants. A toxinmay be generated by a biological material and/or biological agent. Atoxin may be synthetically manufactured. Examples of toxins include, butare not limited to, toxic secondary metabolites produced by organisms,such as bacteria, plant, marine organism, a fungus. A toxin may beartificially synthesized, including by organic and/or inorganicsynthesis. Plant toxins include, but are not limited to, ricin, abrin.Bacterial toxins include endotoxins and exototoxins, such as botulinum,Shiga. Marine toxins include, but are not limited to, saxitoxin,palytoxin. Fungal toxins include, but are not limited to, mycotoxinssuch as aflatoxin, citrinin, fumonisins, ochratoxin A, patulin,trichothecenes, zearalenone, and ergot alkaloids such as ergotamine.

As used herein, “analyte” refers to a volatile component associated withthe biological material, including a microorganism, toxin or, moregenerally, chemical, and that can be used to help identify thecorresponding biological material, including in combination with otheranalytes released by the biological material. The analyte may bereleased by the biological material as a normal part of the metabolicpathway. The analyte may be described as a volatile organic compound(VOC), such as a microbial volatile organic compound (MVOC). The methodsand devices are particularly suited as training aids because they havebeen rendered-safe, and there is no chance of inadvertent unwantedexposure during use, as the only relevant biological material are theVOCs that are not, in and of themselves in the amounts found in thepolymer, dangerous to the environment, user, or the public.

A polymer layer is considered in “proximity” to a material, including abiological material, if volatile organic compounds from the material arecapable of coming into contact with the polymer material, so that thepolymer material becomes charged with the VOC and can be used to latertrain, including canines. The proximate position is preferably not inphysical contact with the biological material. Given the ease by whichVOC diffuse or can be conveyed (e.g., via convection and/or diffusion),the proximity of the polymer to the biological material to achievedesired charge of the polymer with the VOC is rather tolerant, and caninclude separation distances up to 10 cm or greater.

“Air-tight” refers to there being no leakage or communication betweenthe inner volume formed by the canister and lid, and the surroundingenvironment. Of course, the invention is compatible with some minimalleakage, including being described as “substantially” air-tight. Suchleakage is preferred to be minimized, as the VOCs will also tend toleak, thereby decreasing device shelf-life. In this context,“substantially” may refer to air leakage that is selected to have avalue such that the shelf-life of the device is on the order of betweenat least 1-6 months without undue impact on training sensitivity andspecificity.

As used herein, “sensitivity” of the training aid indicates thelikelihood a canine will be alerted in a training assay.

As used herein, “specificity” of the training aid indicates thelikelihood a canine will discriminate the target from a distractor orbackground VOC.

As used herein, “inactivating” refers to a process that can render-safethe training aid so that there is no concern with respect to how thetraining aid is handled, stored, transported or used because there is norisk of secondary infection from the training aid. The inactivating is,functionally, equivalent to a sterilization where potentially infectiousmicroorganisms (bacteria, virus) are inactivated. The methods providedherein are compatible with a range of inactivating processes that removemicroorganisms or capacity to be infective, including, but not limitedto, saturated steam under pressure (e.g., autoclave), hot air,filtration, irradiation (gamma and electron-beam radiation) that do notadversely impact the ability of the polymer to be charged with an odorassociated with the biological agent. See, e.g., The InternationalPharmacopoeia (Ninth Edition, 2019) at 5.8 “Methods of sterilization.”Although gas and chemical inactivation are possible, they are preferablyavoided as they have a tendency to either add new odor that is unwantedor would compromise the polymer layer (e.g., make it brittle).

The methods and devices presented herein are compatible with a range ofmaterials to capture, store and release VOCs associated with infectiousbiological material, including BSAT. For example, a silicone elastomermay be used, including polydimethylsiloxane (PDMS).

PDMS is a non-crystalline, hydrophobic polymer suitable for bothabsorption of VOCs into the polymer and uniform release back into thevapor phase. Canine detection training aids using PDMS are a relativelynew technology for the canine community. Unlike previous alternativetraining aids that have tried to mimic hazardous target odors or renderactual hazardous target material safe, the PDMS methods provided hereincapture, contain, and then release the actual vapor emitted from ahazardous target. This is safer and obviates custody and logisticalcomplications as compared to distributing, handling, and storinghazardous targets in their native form in security-restrictedoperational environments, such as a mass transit system or a publicspace.

PDMS can be polymerized using a two-part base/curing agent mixture andcan be cured with either heat or at room temperature. PDMS can bedispensed directly into the canister in which it is cured. PDMS canwithstand an operational temperature range of −45° C. to 200° C., whichis within the temperature range used to remove VOCs associated with thecanister and for the resulting autoclave sterilization process after thetarget VOCs are added.

FIG. 1 is a representative schematic of a method of making a trainingaid. A biological material 10 emits analytes as indicated by arrows 20.In this example, the biological material 10 is illustrated as cellculture having cell culture media. The cells may be dispersed in themedia or may be positioned on a solid substrate at the bottom of thecell culture media. As described, the analytes may be characterized asVOC or MVOC. The analytes diffuse or are otherwise conveyed to polymer30 positioned proximate to and separated from biological material 10.This “charging” of the material may be carried out in a secure area 5capable of handling dangerous materials. The charged polymer 32 is thenheated to obtain a render-safe polymer 34. The render-safe polymer 34 isalso referred herein as a sterilized polymer, reflecting that theheating step ensures there is no active biological material 10associated with the polymer 34.

The render-safe charged polymer 34 can be stored in an air-tightcanister 40 with removable lid 50. The canister 40 may have a bottomsurface 41, side wall(s) 42, and open upper surface 43 to form an innervolume illustrated as 44 (corresponding to polymer volume plus theair-space between the polymer surface and the facing lid surface). Thecanister has a depth, as illustrated by 45, and the polymer a thickness,as illustrated by arrows 31. Canister depth 45 may correspond to lengthof sidewall(s) 42, such as between about 1 cm and 3 cm, or about 2 cm.Canister may be circular in cross-section, so that there is a singlesidewall, with a diameter between about 1 cm and 30 cm, includingbetween 3 cm and 10 cm, or about 6 cm. A circular cross-section canisteris preferable, although other shapes are compatible, including squares,rectangles, triangles, ellipses and the like. Smooth edges are preferredover edges having corners for better distribution of VOCs that otherwisemay have a tendency to “stick” in the corners.

FIG. 4A illustrates a method that uses an inactivating step to obtainrender-safe charged polymer 34. The inactivating step may correspond toa heating step as described in FIG. 1 or another post-chargedinactivation, such as irradiation or intense light, including exposureto ionizing radiation, UV illumination or other wavelength that caninactivate unwanted biological agents that could cause harm whileensuring the charged polymer remains effective as a training aid.

Alternatively, the inactivating may be prior to charging, as illustratedin FIG. 4B. This may be by filtering to remove unwanted dangerouscomponents from the biological material while the odor-releasingcomponents remain to be used for charging. Again, any inactivation thatselectively inactivates the dangerous (including infectious) components,without adversely impacting the vapor-releasing components useful fortraining, can be used. This could include a heating step. The biologicalmaterial 10 undergoes an inactivation step before charging of polymerlayer 30 to obtain a “safe” biological material 11 that is used tocharge polymer layer 30. Accordingly, a render-safe charged polymer 34can be obtained directly from the charging step. As desired, multipledifferent inactivation steps may be used, including pre and/orpost-charging, for extra safety. For example, a combination offiltering, heating, and irradiation may be used.

FIG. 2 illustrates the training aid in a stored (top panel) and deployed(deployed) configuration. To store the aid, the lid 50 is affixed to thebottom portion 40 of the canister, with the charged and sterilizedpolymer layer contained within the closed volume formed by the lid andbottom canister 40. To use the training aid, the lid 50 is removed,thereby exposing the charged and sterilized polymer to the environmentand corresponding release of the captured VOCs 60 to the surroundingenvironment, for detection by a detector, such as an artificial sensoror an animal (e.g., canine) olfactory system. The dashed lines labeled“apply lid” and “remove lid” reflects that the aid can be re-used,including over relatively lengthy time periods. For applications wherethere is concern of losing lid 50, the aid may incorporate exposablevents, where there is a one-piece canister and by a twisting ortranslation motion, motion of one piece exposes or covers vents or alarge opening on the top or bottom surface of the canister.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1: Polymer Odor Capture and Release (POCR) Design—Preparing thePDMS Layer

Referring to FIGS. 1-2, this example demonstrates polymer odor captureand release (POCR) design contains the following: a canister 40 with alid 50, a polydimethylsiloxane (PDMS) membrane 30 32 34, and microbialvolatile organic compounds (MVOCs) 20 from a biological agent or toxin10.

The PDMS can be provided in a canister and lid configuration. Thecanister and lid are constructed of shallow, seamless, tin plated steelformed from 0.24 mm metal or other material (e.g. glass) able towithstand the sterilization process, such as a pressure and temperaturesterilization process associated with autoclaving. The canister and lidcan be round and described as having a minimum depth of and a minimumdiameter. Examples of a minimum depth and minimum diameter include, butare not limited to, ¾″ and 2.5″, respectively. The canister and lidpreferably do not contain any additional openings in order to containthe target VOCs when not in use. For example, the canister and lidtogether may form a substantially airtight seal, where there is minimalvapor loss from the sealed canister and lid and minimal air entry intothe interior of the canister and lid from the environment surroundingthe canister and lid.

In order to remove inherent and unwanted VOCs from the canisters andlids prior to adding the PDMS membrane and target VOCs, a bakingprocedure is performed. This process removes factory, shipping, curing,and non-target biological odors. With an oven preheated for one hour at150° C., lids are removed from the canisters and both components arebaked at 150° C. for four hours. Once cooled, the lid is placed on thecanister and Teflon® tape can be placed around the edge of each lidduring storage for an additional seal.

Polydimethylsiloxane Membrane: Commercially available grades of PDMS canbe used. For example, SYLGARD′ 184 (Dow Corning SKU 2065622) can beused.

PDMS is prepared according to manufacturer instructions and poured intobaked, cooled canisters to a depth of at least 2 mm. SYLGARD™ 184includes a volatile impurity, ethylbenzene. In order to remove thisimpurity from the polymeric substrate, the VOCs from this impurity canbe removed through a second baking process. Once cured, and with thelids removed, the canisters with PDMS and the lids are baked at 150° C.for two hours. Once cooled, the lid is placed on the canister andTeflon® tape is placed around the edge of each lid for storage.

Example 2: Polymer Odor Capture and Release (POCR) Design—Loading thePDMS Layer

Target Biological Agent MVOCs: During metabolism, microorganisms produceMVOCs. PDMS can be infused with MVOCs by suspending the PDMS-containingcanister over a biological agent for a period of days to weeks. Thisindirect method of capturing MVOCs minimizes microbial contamination ofthe POCR.

Example 3: Polymer Odor Capture and Release (POCR) Design—Sterilizingthe PDMS Layer

POCRs infused with VOCs are sterilized, such as by autoclaving at 121°C. for 60 minutes. This method of steam sterilization is common practicein microbiological laboratories and is effective at combining fourparameters to quickly kill microorganisms: steam, pressure, temperature,and time. After autoclaving, the responsible laboratory conducts dailysterility checks for an agent-specific period of time, typically up toseven days, before a laboratory can confirm the POCRs non-hazardous andnon-infectious so that they may be safely shipped and used for caninedetection training in the field.

Example 4: Experimental Results

Bovine Viral Diarrheal Virus (BVDV)

Trial design: Five canines are used in all trials described below anddetection is performed using a six-arm scent wheel. Distractors includea closely related bovine virus (bovine herpesvirus 1 Colorado strain),prepared cell culture media, and the following components of cellculture media: purified water, minimal essential media with Earle'ssalts, L-glutamine, an antibiotic combination consisting ofpenicillin/streptomycin/amphotericin, sodium bicarbonate, and equineserum. Canines are operated off-lead by a handler, who was blinded tothe target location.

Autoclaved POCR-BVDV: Canines are presented 57 autoclaved POCR-BVDVtargets and 612 distractors. Canines alert to 52 of 57 autoclavedPOCR-BVDV targets and alert to 8 of 612 distractor presentations. Thesensitivity of canine detection is 91.2% (95% confidence interval [CI],81.0, 96.2), indicating a high capability to identify the target usingthe scent wheel. The canines' specificity is 98.7% (95% CI, 99.4, 97.4)indicating a high capability to discriminate the target virus from thedistractors (Table 1).

Live BVDV Cell Culture:

Canines are presented 49 live cell cultures of BVDV and 327 distractorpresentations. Canines alert to 49 of 49 live BVDV cell culture targetsand alert to 1 of 327 distractor presentations. The sensitivity ofcanine detection is 100% (95% CI, 92.7, 100), indicating a highcapability to identify the target. The canines' specificity is 99.7%(95% CI, 98.3, 99.9), indicating they were capable of discriminating thetarget virus from the distractors (Table 1).

Bacillus anthracis

Trial design: Six canines are used in trials described below anddetection is performed using simulated operational searches of buildingsor a six-arm scent wheel. Distractors include two strains of Bacilluscereus, two strains of B. thuringiensis, blank nutrient agar plates, andthe individual components of the nutrient agar (beef extract, peptone,agar). Blank POCRs are placed throughout the laboratory to absorb otherlaboratory-associated odors, serving as an additional distractor.Canines are operated off-lead by a handler, who is blinded to the targetlocation.

Selection of distractor B. cereus and B. thuringiensis strains: B.anthracis, B. cereus, and B. thuringiensis are closely related speciesbelonging to the Bacillus cereus sensu lato group. Because there is morediversity amongst strains of B. cereus and B. thuringiensis, for eachspecies we select one strain more closely related to B. anthracis andone less related, for a total of four distractor strains. (FIG. 3)

Selection of B. anthracis strains: Five strains of B. anthracis wereincluded: Sterne strain and four cured B. anthracis strains provided bythe U.S. CDC (FIG. 3).

Autoclaved POCR-B. anthracis Sterne Strain (POCR-BA Sterne):

Canines are presented 120 autoclaved POCR-BA Sterne targets and 220autoclaved distractor presentations hidden for simulated operationalsearches of buildings. The purpose of this part of the experiment is todetermine if canines could discriminate between B. anthracis and closelyrelated species. As B. cereus is a common microorganism found in soiland B. thuringiensis is a natural biological pesticide used inagriculture, this is also an important operational consideration ascanines are likely to encounter both species in their environment on aregular basis. One of the B. thuringiensis strains selected is isolatedfrom Thuricide®, a widely used pesticide in the United States.

Canines alert to 91.7% (95% CI, 85, 95) of autoclaved POCR-BA Sternetargets and alert to 16% (95% CI, 12, 22) of autoclaved distractorpresentations. This is an unusually high false positive rate, attributedto the autoclaving process contaminating samples with a native scentthat caused targets and distractors to share a common set of odors. Thisis furthered by the fact that canines have difficulty discriminating alldistractors, not just distracting bacteria. Further testing is conductedon a six-arm scent wheel using POCRs containing autoclaved (n=220) andunautoclaved (n=402) distractors. Canines alert to 16% (95% CI, 9, 26)of autoclaved distractors and 3.7% (95% CI, 2.2, 6) of unautoclaveddistractors. To mitigate the high false positive rate, new and dedicatedautoclaves for distractors may be utilized.

With a sensitivity of 91.7% (95% CI, 85, 95), canine detection resultsindicate a high capability to identify the target in an operationalenvironment. The canines' specificity on unautoclaved distractors isalso high, indicating a high capability to discriminate the target virusfrom the distractors, including other strains of Bacillus spp.

Live Bacillus anthracis Cultures: A six-arm scent wheel is used todetermine whether canines trained on POCR-BA Sterne are able togeneralize to four cured strains of B. anthracis. Canines are presented332 live BA cultures. Canines give a positive indication on 91.2% (95%CI, 87, 93) of all five of the BA targets. Also noteworthy, canines givea positive indication 91.6% (95% CI, 74, 97) the very first time theyexperienced the four new cured BA targets (n=24), indicating the caninesare able to generalize from BA Sterne to other BA strains. Canines givea false indication on 0.4% (95% CI, 0.1, 1) of live distractors (n=996)during trials that had a positive target present. Canines give a falseindication on 3.7% (95% CI, 2, 4) of distractors (n=1482) during trialsduring which a target is not present.

Example 5: SARS-CoV-2 (Causative Agent of COVID-19)

An example of use of the training aid for detection of an emergingthreat is for detection of a biological agent that is causing or likelyto cause a pandemic, including a coronavirus such as the one that causesCOVID-19. Exemplary methods include culturing cells capable of beinginfected by the virus known as severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019(COVID-19). For example, the virus has been cultured with Vero-CCL81cells and Vero E6 cells. Harcourt J, et al. Severe Acute RespiratorySyndrome Coronavirus 2 from Patient with Coronavirus Disease, UnitedStates. Emerg Infect Dis. 2020; 26(6):1266-1273.https://dx.doi.org/10.3201/eid2606.200516. Other cell lines include ahuman-derived cell line, such as Huh7 cells. The polymer layer may beexposed directly to the cell culture or may be exposed to cell culturemedia removed from the cell culture, thereby capturing one or moreanalytes and obtaining an analyte-infused polymer layer associated withSARS-CoV-2. The removed cell culture material may be filtered prior toexposure to the polymer layer. The analyte-infused polymer layer may besubject to inactivation, including by steam heating under pressure, toensure the training aid is rendered safe for subsequent use. Therender-safe analyte-infused polymer layer is then stored in asubstantially air-tight canister with a removable lid for subsequent usein training.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

TABLE 1 Comparison of canine detection of autoclaved BVDV targets versuslive BVDV targets. Canines are presented 57 autoclaved POCR-BVDV targetsand 612 distractor presentations. Canines alert to 52 of 57 autoclavedPOCR-BVDV targets and alert to 8 of 612 distractor presentations. Thesensitivity of canine detection is 91.2% (95% confidence interval [CI],81.0, 96.2), indicating a high capability to identify the target usingthe scent wheel. The canines' specificity is 98.7% (95% CI, 99.4, 97.4),indicating a high capability to discriminate the target virus from thedistractors Live BVDV culture Autoclaved POCR-BVDV (high infection) #Targets 57 49 # Target 52 49 alerts # Distractors 612 327 # Distractor 81 alerts Sensitivity 91.2% (95% CI, 81.0, 96.2) 100% (95% CI, 92.7, 100)Specificity 98.7% (95% CI, 99.4, 97.4) 99.7% (95% CI, 98.3, 99.9)

1. A method of making a training aid for detecting a biohazard, themethod comprising the steps of: providing a material that releases oneor more analytes; positioning a polymer layer in proximity andphysically separated from the material, wherein the one or more analytesare capable of release from the material and contact with the polymerlayer; capturing the one or more analytes that contact the polymer layerto obtain an analyte-infused polymer layer; inactivating any agents onor in the analyte-infused polymer layer to render-safe theanalyte-infused polymer layer; and storing the render-safeanalyte-infused polymer layer in a substantially air-tight canister witha removable lid for subsequent use in training; thereby making thetraining aid for biohazard detection.
 2. The method of claim 1, whereinthe material comprises a biological material and the agent is abiological agent.
 3. The method of claim 2, wherein the inactivatingstep comprises heating the analyte-infused polymer layer to atemperature sufficient to inactivate biological agents on or in theanalyte-infused polymer layer.
 4. The method of claim 1, wherein theinactivating step is selected from the group consisting of: steamheating; dry heating; irradiating with electromagnetic radiation; andfiltering the material prior to the capturing step to remove anybiological materials and prevent biological materials from contactingthe polymer layer.
 5. The method of claim 1, wherein the polymer layeris positioned in an inner volume defined by a shallow and seamlesscanister.
 6. The method of claim 5, wherein the removable lid isconfigured to cover the canister inner volume having the polymer layerin a substantially air-tight configuration.
 7. The method of claim 1,further comprising the step of heating the polymer layer and a canisterbefore the positioning step to remove unwanted volatile organiccompounds associated with the polymer or the canister.
 8. The method ofclaim 7, wherein the heating step comprises heating the analyte-infusedpolymer layer and canister.
 9. The method of claim 1, wherein theinactivating step comprises: autoclaving the polymer layer to anelevated temperature and pressure to sterilize the analyte-infusedpolymer layer.
 10. The method of claim 9, wherein the autoclavingfurther comprises steam-sterilization of the analyte-infused polymerlayer.
 11. The method claim 2, wherein the inactivating step comprisesintroducing the analyte-infused polymer layer to a temperature that isbetween 125° C. and 155° C. for a time period of between 0.5 hours and1.5 hours.
 12. The method of claim 11, wherein after the heating andstoring step, sufficient analyte remains in the analyte-infused polymerlayer such that the training aid used to train a canine to detect thebiological agent has a sensitivity of at least 85% and/or a specificityof at least 90%.
 13. The method of claim 5, wherein prior to the polymerlayer positioned in the inner volume the method further comprises thesteps of: separating the lid from the canister; baking the separated lidand canister at 140° C.-160° C. for at least three hours; cooling theseparated lid and canister; and sealing the lid to the canister untilthe canister is ready to receive a pre-polymer or the polymer layer. 14.The method of claim 1, wherein the polymer layer comprisespolydimethylsiloxane (PDMS).
 15. The method of claim 14, wherein thePDMS layer has a depth that is greater than or equal to 2 mm and lessthan or equal to a canister inner volume depth.
 16. The method of claim1, further comprising the step of removing unwanted polymer impuritiesby heating the polymer layer to at least 125° C. for at least one hour.17. The method of claim 1, further comprising the steps of: providing atleast one microorganism and/or toxin that releases an analyte comprisinga volatile organic compound (VOC); and suspending the polymer layer overthe biological agent for a time period sufficient to contact thereleased VOC with the polymer layer; thereby loading the polymer withthe VOC.
 18. The method of claim 17, wherein the microorganism isselected from the group consisting of: a bacteria; a virus; a fungus; aprion; and a combination thereof.
 19. The method of claim 1, wherein:the material comprises a toxin, the toxin selected from the groupconsisting of: a plant toxin; a bacterial toxin; a marine toxin; afungal toxin; and any combination thereof; and the inactivating stepcomprises autoclaving and/or filtering.
 20. The method of claim 1,wherein the lid and canister in a stored configuration provides ashelf-life for a canine-training application of at least six-months. 21.A method of training a canine, the method comprising the steps of:providing a training aid comprising: a canister having a bottom surfaceand one or more side walls to define an interior volume with an openupper surface; a sterilized volatile organic compound (VOC)-infusedpolymer positioned supported by the canister bottom surface; and aremovable lid removably connected to the canister one or more side wallsto contain the VOC-infused polymer in the interior volume; removing theremovable lid from the canister one or more side walls; exposing thecanine to a volatile organic compound released from the VOC-infusedpolymer supported by the canister bottom surface; thereby training thecanine.
 22. The method of claim 21, wherein the training aid isreusable, the method further comprising the step of: reusing therender-safe training aid at least 10 times for the same canine, adifferent canine, or a combination of the same and different canines.23. A render-safe training aid comprising: a canister having a bottomsurface and one or more side walls to define an interior volume with anopen upper surface; a removable lid removably connected to the canisterone or more side walls; and a sterilized volatile organic compound(VOC)-infused polymer positioned in the interior volume and supported bythe canister bottom surface.